The principal objective of this proposal is the elucidation of the specific biochemical mechanisms at the molecular level that are associated with the orderly control of vitamin D3 metabolism. Two systematic approaches are described. First, current efforts will continue on the isolation and characterization of specific protein components functional in the renal and hepatic hydroxylases of vitamin D3 and 25-hydroxyvitamin D3 (25-OH-D3). These components are primarily the P-450 cytochromes of the renal 1 alpha-and 24-hydroxylases, and their respective reductases. Additional characterization work involves the hepatic cytosol 25-OH-D3 binding protein. The role of this protein in the reactions catalyzed by the hepatic 25-hydroxylase will be studied. Antibodies in rabbits will be produced against the hepatic binding protein for the evaluation of its relationship to kidney cytosol and plasma 25-OH-D3 binding proteins. Functional comparisons between mitochondrial and microsomal 25-OH-D3 metabolizing enzymes will be made to define the interrelationships among cell organelles responsible for the regulation of cellular 25-OH-D3 concentrations. Work concerning the biogenesis of mitochondria and the synthesis of the 1 alpha-and 24-hydroxylases will involve studies on the rate of incorporation of radioactive amino acids into apoproteins and of 59Fe into heme. These studies will be extended to test the regulation of the l alpha-hydroxylase activity by phosphorylation. Specific incorporation of 32P into kidney mitochondrial cytochrome P-450 will be explored. Correlations will be sought between the rates of this incorporation and of the appearance of 1 alpha-hydroxylase activity in isolated mitochondria. The induction of a specific 1 alpha-hydroxylase phosphatase as a mechanism for the inactivation of the 1 alpha-hydroxylase will be examined. These studies will form the basis for describing the regulation of the integrated endocrine system associated with the metabolism of vitamin D3.